Summary

The retinotectal projection is a premier model system for the investigation
of molecular mechanisms that underlie axon pathfinding and map formation.
Other important features, such as the laminar targeting of retinal axons, the
control of axon fasciculation and the intrinsic organization of the tectal
neuropil, have been less accessible to investigation. In order to visualize
these processes in vivo, we generated a transgenic zebrafish line expressing
membrane-targeted GFP under control of the brn3c promoter/enhancer.
The GFP reporter labels a distinct subset of retinal ganglion cells (RGCs),
which project mainly into one of the four retinorecipient layers of the tectum
and into a small subset of the extratectal arborization fields. In this
transgenic line, we carried out an ENU-mutagenesis screen by scoring live
zebrafish larvae for anatomical phenotypes. Thirteen recessive mutations in 12
genes were discovered. In one mutant, ddl, the majority of RGCs fail
to differentiate. Three of the mutations, vrt, late and
tard, delay the orderly ingrowth of retinal axons into the tectum.
Two alleles of drg disrupt the layer-specific targeting of retinal
axons. Three genes, fuzz, beyo and brek, are
required for confinement of the tectal neuropil. Fasciculation within the
optic tract and adhesion within the tectal neuropil are regulated by
vrt, coma, bluk, clew and blin.
The mutated genes are predicted to encode molecules essential for building the
intricate neural architecture of the visual system.

Most of the molecular players listed above have been identified through
biochemical purification or by candidate gene approaches. A forward-genetic
screen provides an alternative strategy to reveal novel genes (or new
functions for known genes) in an unbiased manner. A previous anatomical screen
for retinotectal projection defects in zebrafish used fluorescent lipophilic
dyes to trace RGC axons as they navigated to the tectum. Two carbocyanine
dyes, DiI and DiO, were injected at different locations into the retina of
larval zebrafish, thus labeling separate subpopulations of RGCs terminating in
topographically distinct regions of the tectum
(Baier et al., 1996;
Karlstrom et al., 1996;
Trowe et al., 1996). This
large-scale screen uncovered 114 mutations, in about 35 genes, disrupting
either the pathfinding of axons from the eye to the tectum or the retinotopic
map. Although some of the more specific mutations, such as gna, woe
and nev, still await molecular identification, most of the genes have
now been cloned (Culverwell and Karlstrom,
2002). Unsurprisingly, retinotectal pathfinding was found to
depend on proper brain patterning (Hedgehog signaling, syu, igu, con, dtr,
uml and yot; Nodal signaling, cyc; homeodomain
transcription factors, noi) and on components of the extracellular
matrix (bal, gpy and sly). A small minority of mutations was
found to disrupt specific signaling pathways within the retinal growth cones,
such as those mediated by Slit/Robo (ast)
(Fricke et al., 2001), heparan
sulfate proteoglycans (box and dak)
(Lee et al., 2004) or
PAM/highwire (esr) (D'Souza et
al., 2005).

Although productive with regard to isolating retinotectal mutants, this
first screen was laborious and intrinsically limited to finding only a subset
of interesting phenotypes. Important organizing principles of retinotectal
connectivity, such as neuropil assembly and laminar targeting of RGC axons
could not be investigated with the screening method employed. As a
consequence, the factors that assemble the characteristic architecture of
optic tract and tectum and coordinate the development of the visual system are
still elusive. We expect that disruption of some of these factors may lead to
relatively subtle anatomical alterations, whose detection and analysis require
sensitive methods. As structural changes of the CNS will ultimately influence
its function, mutations in the human homologs of these genes might turn out to
be responsible for neurological and psychiatric diseases.

We have developed a screening strategy aimed at discovering specific
disruptions of retinotectal architecture. Enhancer sequences from the
zebrafish brn3c gene were used to drive membrane-targeted GFP in a
distinct subset of RGCs. GFP-based forward-genetic screens have been employed
extensively in Drosophila and C. elegans, and more recently
in zebrafish (Lawson et al.,
2003). The use of a genetically encoded label overcomes the
limitations of dye injections. First, the screening assay is rapid, because
fish do not need to be aldehyde fixed and injected. Second, the labeling is
robust and reproducible among fish, thus allowing the detection of even subtle
abnormalities. Finally, the GFP label allows the observation of the same fish
at multiple stages of development. We tested the suitability of the
Brn3c:mGFP transgenic line in a screen of 233 ENU-mutagenized F2
families. Our relatively small-scale effort (three investigators, 1 year)
detected 13 novel phenotypes, including ones in which the retinotectal
projection is delayed or disorganized. These new mutants should add to a
cellular and molecular understanding of the processes that generate precise
neuronal connections in the visual system and other parts of the nervous
system.

Materials and methods

Fish breeding

Zebrafish of the TL strain were raised and bred at 28.5°C on a 14 h
light/10 h dark cycle. Embryos were produced by natural crosses and staged by
hours or days post fertilization (hpf or dpf).

Cloning of zebrafish brn3b and brn3c

A 168 bp brn3b fragment was cloned from zebrafish cDNA by
degenerate PCR. Further parts of the sequence were obtained using degenerate
primers targeted to conserved regions at the 5′ and 3′ ends of the
brn3b sequence. At least two clones from independent PCR reactions
were sequenced. Primers to the resulting consensus sequence of these fragments
were used to identify genomic PAC clones in a pooled PAC library available
from RZPD (Berlin, Germany) (Amemiya and
Zon, 1999). Three clones contained brn3b. The remaining
brn3b sequence was obtained by direct sequencing from PAC clones
BUSMP706A1597Q2 and BUSMP706N19174Q2 from RZPD. The GenBank Accession Number
for brn3b is AF395831.

Zebrafish full-length brn3c was identified as for brn3b,
except that primers for nested PCR were designed based on a partial cDNA
sequence published previously (Sampath and
Stuart, 1996). The GenBank Accession Numbers for full-length
brn3c and its first intron are AY995217 and AY995218. The forward
primers were 5′-GGC AAT ATA TTC AGC GGC TTT G-3′ and 5′-GCT
AAA CTC CTC GTA TTG TTA C-3′. The reverse primers were 5′-GTA TCT
TCA GGT TGG CGA GAG-3′ and 5′-GGA GGA AAT GTG GTC GAG
TAG-3′. Three positive PAC clones were identified.

Construction of the Brn3c:mGFP transgenic vector

The brn3c-containing PACs were characterized by restriction
digests and Southern hybridization. A 7.5 kb BspEI fragment was
identified that contained part of the brn3c-coding region and 6 kb of
upstream sequence. It was subcloned from PAC clone BUSMP706K02247Q2 and
partially sequenced. The PAC sequences, together with a 5′ RACE product,
yielded the remaining parts of the zebrafish brn3c sequence and
identified the putative translation start. The 6 kb promoter fragment was
cloned into pG1, a GFP expression vector (kindly provided by C.-B. Chien, MPI
Tübingen). For membrane targeting, the sequence encoding the first 20
amino acids of zebrafish GAP43 (Kay et
al., 2004) was generated from two overlapping oligonucleotides,
which also contained a Xenopus β-globin ribosomal binding site.
The sequences were 5′-G GAA TTC CAC GAA ACC ATG CTG TGC TGC ATC AGA AGA
ACT AAA CCG GTT GAG AAG-3′ and 5′-TCC CCC GGG CTG CAG CTG ATC GGA
CTC TTC ATT CTT CTC AAC CGG TTT AGT-3′. The oligonucleotides were fused,
filled in with Klenow Polymerase, and cloned into the PstI and
EcoRI site of the brn3c promoter. The resulting pTR56 vector
was used to generate transgenic zebrafish (see below).

Generation of transgenic fish

The insert from vector pTR56 was excised by digestion with NotI
and separated from the vector backbone by agarose gel electrophoresis. The DNA
was extracted and eluted in 10 mM triethanolamine (Tris, pH 7.5). Prior to
injection, the DNA was diluted in water containing 0.05% Phenol Red (Sigma) to
10-20 ng/μl. Injected embryos were raised to sexual maturity and crossed to
identify founder fish. The embryos from these crosses were scored for their
GFP expression and raised. This effort led to the production of several stable
lines, one of which was used in this study. The official designation of this
line is TG(Brn3c:GAP43-GFP)s356t
(http://www.zfin.org).

Mutagenesis

To efficiently induce random point mutations in the zebrafish genome, we
followed published protocols (van Eeden et
al., 1999). Briefly, adult male TL fish were treated three to five
times at weekly intervals with ENU (3.0 mM, 1 hour). Four weeks after the last
treatment, they were outcrossed to produce up to 200 F1 fish per male. These
F1 fish were crossed to Brn3c:mGFPs356t carriers to
generate F2 families. Six or more pairs of random crosses were set up between
siblings for each F2 family. In total, 233 F2 families were screened.

Screening assay

Embryos (3 dpf) and larvae (6 dpf) were embedded in 2.5% methylcellulose in
E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 33 mM
MgSO4) and screened under a Leica MZ25 fluorescence-equipped
dissecting microscope with 100× magnification. Embryos with obvious
morphological defects prior to 3 dpf were discarded. We scored the presence of
retinal fibers in the tectum (particularly at 3 dpf), the trajectory of
GFP-positive fibers in the optic tract, the width and shape of the optic
tract, the density of tectal innervation, the size and shape of the tectal
neuropil, the layer structure of the tectum, and the appearance of the RGC
axonal meshwork in the tectal neuropil. Crosses in which a quarter of the F3
progeny showed a mutant phenotype were repeated. Two or three such re-tests
were carried out before a mutant was considered a real candidate. In these
cases, one or both of their parents were outcrossed to unrelated
Brn3c:mGFP carriers to establish a mutant stock. The mutation was
recovered in the next generation through random pairwise crosses. All mutants
described in this report have been propagated as heterozygous stocks for at
least three generations.

Labeling of the retinotectal projection with carbocyanine dyes

Fish were deeply anesthetized in 0.016% tricaine (Sigma) and fixed in 4%
PFA in PBS for 2-24 hours. They were embedded in 1% agarose in 0.5× PBS)
and pressure injected with dye solution using a PV-820 pressure injector
(World Precision Instruments, Sarasota, FL). The carbocyanine dye DiI
(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlorate, Molecular Probes D-282) was dissolved either in
N,N-dimethylformamide (DMF) for fixed larvae or in ethanol for live larvae to
a concentration of 2% (m/v). DiO
(1,1′-dioctadecyl-3,3,3′,3′-tetramethyloxacarbocyanine
perchlorate, Molecular Probes D-275) was dissolved in chloroform (2% m/v). To
visualize the retinotectal map, DiI and/or DiO were injected into the eyes of
larvae and imaged 12-24 hours later.

Confocal imaging

Larvae were embedded in a 0.5×20 mm imaging chamber (CoverWell, Grace
Bio-labs) in 1.2% low melting point agarose dissolved in E3 medium containing
0.8% norepinephrine and 0.016% tricaine. For immunohistochemistry, the left
eye was removed and stained embryos/larvae were mounted laterally in 70%
glycerol in PBS. Confocal image stacks were acquired using either a BioRad
1024M or a Zeiss 510 META laser-scanning microscope. Long working distance
objectives, 20× (air, NA 0.5) and 40× (water, NA 0.8), were used.
To reconstruct axons and their arbors, a series of optical planes were
collected (z-stack) and collapsed into a single image (maximum
intensity projection) or rendered in three dimensions to provide views of the
image stack at different angles. The step size for each z-stack was
chosen upon calculation of the theoretical z-resolution of the
objective used (typically 0.5-1 μm).

Results

Regulatory regions of brn3c drive stable expression of GFP
in a subset of RGCs

We identified zebrafish brn3c (brn3.1, pou4f3) by PCR
cloning. The putative amino acid sequence of Brn3c consists of 331 amino
acids. Compared to mouse Brn3c, 81% of the amino acid positions are identical
within the total sequence and 97% within the POU-domain. We confirmed the
annotation by also cloning a highly related gene, brn3b (brn3.2,
pou4f2) (DeCarvalho et al.,
2004). A phylogenetic tree based on ClustalW sequence comparison
(Higgins and Sharp, 1988)
places the zebrafish Brn3b and Brn3c sequences into separate clades together
with their mammalian orthologs (data not shown).

A large subset of RGCs express GFP in the stable Brn3c:mGFP
transgenic line. (A) Schematic drawing of the DNA construct used to generate
the Brn3c:mGFP transgenic line. (B) Lateral view of 56 hpf live
Brn3c:mGFP transgenic embryo showing GFP expression by RGCs and
mechanosensory hair cells (neuromasts) of the lateral line and inner ear. The
optic nerve is visible. (C-E) Fixed Brn3c:mGFP transgenic embryos
labeled by whole-mount immunohistochemistry. (C) Ventral view of a 42 hpf
retina, labeled with anti-GFP. Anterior (nasal) is upwards. GFP expression
starts in a small cluster of cells in the ventronasal retina, near the optic
fissure. The arrowhead indicates the first GFP-positive axons exiting the eye
through the optic stalk. (D) Lateral view of a 44 hpf retina, labeled with
anti-GFP in green and zn5 in red. GFP has spread into central retina. The
onset of Brn3c:mGFP transgene expression follows that of zn5 by∼
6 hours. (E) Lateral view of a 72 hpf retina, labeled with anti-GFP.
GFP-positive RGCs are distributed uniformly throughout the ganglion cell
layer. Scale bars: 200 μm in B; 20 μm in C-E.

To generate a stable transgenic zebrafish line able to drive GFP expression
in RGCs, we isolated about 6 kb of genomic sequence upstream of the
brn3c-coding region and cloned it into a GFP expression vector
(Fig. 1A) (see Materials and
methods). Enhanced GFP, fused to the first 20 amino acids of GAP43, served as
the marker gene, as described previously
(Kay et al., 2004). The GAP43
sequence provides a membrane-targeting signal and ensures complete labeling of
neurites (Zuber et al., 1989).
Injection of the construct at the one-cell stage resulted in transient
expression in select populations of neurons. Fish that showed bright
expression in a large number of cells were raised to adulthood. Eighty-four
injected fish were outcrossed against wild-type fish and analyzed for germline
transmission. A total of 10 germline transgenic founder fish (12% of the
injected embryos) were identified by visual inspection of their progeny under
a fluorescence-equipped dissecting microscope. Offspring of these founder fish
were raised to establish lines. The experiments described here were carried
out in transgenic line TG(Brn3c:mGFP)s356t.

The Brn3c:mGFP transgenic line reveals the architecture of the
retinotectal projection. (A-C) Lateral view of the retinofugal projection in a
6 dpf fixed larva, whose eye has been removed (projection of a confocal
z-stack). Specimen was only lightly fixed to preserve GFP label. (A)
Brn3c:mGFP transgene expression. Four arborization fields, AF-6,
AF-7, AF-8 and tectum (AF-10), are visible, as well as neuromasts of the
lateral line. (B) Retinofugal projection, labeled with DiI following
intraocular injection. (C) Merged view of the two labels shown in A and B. (D)
Transverse section of 7 dpf Brn3c:mGFP transgenic larva, showing
AF-7, AF-8 and the tectal layers. (E-G) Optical sections of tecta in 6 dpf
live larvae (dorsal view; anterior is upwards, midline is towards the right).
(E) Shh:GFP labels all four retinorecipient layers in the tectum. (F)
Brn3c:mGFP labels the SO and SFGS. (G) Double-labeling in vivo with
DiI and Brn3c:mGFP, showing the absence of GFP in one of the three
SFGS sublaminae. For technical reasons, live DiI staining is always
incomplete. As a result, some GFP-only (green) fibers are seen in the SFGS.
(H) Summary of the Brn3c:mGFP labeling pattern. RGCs project to ten
AFs. The largest AF, the tectum, has four layers. Only the green areas receive
significant Brn3c:mGFP input. Red areas are devoid of GFP label.
Yellow area (SO) is innervated mainly by non-GFP fibers, but also receives
weak Brn3:mGFP innervation. AF, arborization field; SO, stratum opticum; SFGS,
stratum fibrosum et griseum superficiale; SGC, stratum griseum centrale; SAC,
stratum album centrale; SPV, stratum periventriculare; asterisks, melanophores
in the skin. Scale bars: 50 μm in A-D; 20 μm in E-G.

All lines showed identical expression patterns, although positional effects
on the levels of GFP expression were observed. At all stages, GFP is
restricted to RGCs and to mechanosensory hair cells of the inner ear and
lateral line neuromasts (Fig.
1B). The earliest GFP expression is observed at about 27 hpf in
the inner ear. At about 42 hpf, GFP expression appears in a ventronasal patch
of RGCs (Fig. 1C). In the hours
that follow, the expression spreads over the entire extent of the retina,
always restricted to a subset of RGCs and their processes and trailing the
expression of DM-GRASP/neurolin, the epitope recognized by the zn5 antibody,
by a few hours (Fig. 1C,D). The
GFP-labeled cells are uniformly distributed within the ganglion cell layer
(GCL) (Fig. 1E) and their axons
approach the optic nerve head in multiple, distinct fascicles. At 5 dpf,∼
50% of the RGCs are GFP positive, while 100% of them are labeled with
zn5. The optic tract and the tectal neuropil are clearly demarcated
(Fig. 2A,B). The label is
stable to adulthood (not shown).

We next asked if the Brn3c:mGFP transgene could be used to
visualize the architecture of the retinotectal projection in zebrafish larvae.
RGCs project axons to ten target areas in zebrafish, of which the tectum is
the largest. These areas have been referred to as retinal arborization fields
(AFs) and are numbered according to their proximodistal position along the
optic tract (Burrill and Easter,
1994). The tectum was named AF-10, being the most distal
retinorecipient area. Brn3c:mGFP-expressing axons were found to
strongly innervate the tectum and, more sparsely, AF-6, AF-7 and AF-8
(Fig. 2A,D). GFP-labeled fibers
are almost completely absent from AF-4, AF-5 and AF-9, and were not detected
in AF-1, AF-2 and AF-3 (Fig.
2A-C; data not shown).

In the tectum of adult cyprinids, four major retinorecipient layers have
been described: the stratum opticum (SO), the stratum fibrosum et
griseum superficiale (SFGS), the stratum griseum centrale (SGC)
and the boundary zone between stratum album centrale and stratum
periventriculare (SAC/SPV) (Meek,
1983; Vanegas and Ito,
1983; von Bartheld and Meyer,
1987). Whole-eye DiI fills reveal that these four retinorecipient
layers are already present in 6 dpf larvae. The same four layers are seen in a
Shh:GFP transgenic line (Neumann
and Nuesslein-Volhard, 2000), in which many RGC axons are
unselectively labeled (Roeser and Baier,
2003) (Fig. 2E).
The most superficial layer, the SO, is about 10 μm thick, densely labeled
and begins at the rostral pole of the tectum. The layer below, the SFGS, is 30μ
m thick and is divided into three sublaminae. The two deeper layers, SGC
and SAC/SPV, are more sparsely innervated. At 6 dpf, these two layers extend
further caudally than the two superficial layers. In zebrafish larvae, unlike
birds and mammals, axons enter their target layer right at the entrance of the
tectum; only a subset of the fibers projects to the SO and these are not seen
`diving down' into the tectum terminating in the deeper layers (data not
shown).

In contrast to Shh:GFP and DiI fills, the vast majority of
Brn3c:mGFP-labeled axons project to the SFGS, and here only to the
two deeper sublaminae (Fig.
2F). The projection to SO is very sparse. The paucity of
Brn3:mGFP axons in SO suggests that only a small subset of the axons
in SO originate from Brn3c:mGFP-expressing RGCs
(Fig. 2G). SGC and SAC/SPV do
not receive Brn3c:mGFP-labeled input. In summary, these labeling
studies show that the Brn3c:mGFP transgene labels a distinct
subpopulation of RGCs with restricted target specificity
(Fig. 2H). Therefore,
Brn3c:mGFP reveals details of the retinotectal architecture that
would be masked by an unselective RGC stain. We employed this line next in a
small-scale screen in search of new retinotectal mutants.

Overview of the screen

For the screen, we mutagenized the spermatogonia of 17 adult male zebrafish
with ENU and outcrossed them to non-mutagenized females. The mutation rate was
determined to be about 0.3% per gene per haploid genome. We used the
pigmentation gene sandy (tyrosinase) as the specific locus for its
easily detectable phenotype (Page-McCaw et
al., 2004). The F1 fish were then crossed to Brn3c:mGFP
carriers to generate 233 F2 families. A total of 1303 crosses were screened by
visual inspection of the GFP-labeled retinotectal projection. Based on the
varying number of crosses per individual F2 family (average 5.6, corresponding
to 0.8 genomes), we calculated that our screen encompassed 168 genomes
(Mullins et al., 1994).

The screen was carried out on F3 progeny at 3 dpf and again at 6 dpf. These
two developmental stages are particularly informative for revealing
perturbations in the retinotectal projection. In wild type, RGC axons reach
the anteroventral boundary of the tectum at 48 hpf. One day later, at 72 hpf
(3 dpf), RGC axon arbors reach the posterodorsal boundary of the tectum and
completely cover the tectum (Stuermer,
1988). Thus, screening at 3 dpf enabled us to uncover mutations
disrupting the initial stages of the retinotectal projection. Between 3 dpf
and 6 dpf, RGC axonal arbors elaborate synaptic connections with tectal
neurons. Screening at 6 dpf thus allowed us to discover mutations affecting
the refinement and maintenance of the retinotectal projection.

Progeny of crosses from 65 F2 families initially showed a putative mutant
phenotype and were re-screened by mating the same pair and scoring their F3
progeny again. Less than one third of these (21) were confirmed and outcrossed
to Brn3c:mGFP transgenic wild type. All but two mutants were
recovered in the next generation. However, six were considered unspecific upon
closer inspection and discarded. The discarded mutants had smaller tecta or
smaller eyes, owing to degeneration or early developmental problems. All
mutants were sectioned at 6 dpf and examined histologically using DAPI to
highlight cytoarchitecture of retina and tectum, in conjunction with anti-GFP.
Two mutations with similar phenotypes were found to be alleles of the same
gene by complementation analysis. As described below, we discovered 13
mutations in 12 genes, which have been grouped into five classes
(Table 1). All mutants are
recessive, completely penetrant with respect to their axon phenotype and are
transmitted in Mendelian ratios. Their phenotypes uncover largely unexplored
processes in the assembly of neural architecture.

The daredevil (ddl) gene is important for the
differentiation of most RGCs

We identified a mutant, ddl, showing a severe reduction of retinal
axons that innervate the tectum at 3 dpf
(Fig. 3A,B). The optic tract is
thin, and RGC axons do not cover the entire tectum. Closer investigation
revealed that the primary defect of this mutant is in generating RGCs. Between
42 and 48 hpf, when newly differentiating RGCs begin to express
Brn3c:mGFP in wild type, GFP expression in the ddl retina is
sparse. This is in contrast to GFP expression in hair cells, which is
comparable with wild type in strength and time of onset (data not shown).
After 60 hpf, when wild-type retina is uniformly filled with GFP-positive
RGCs, the ddl retina contains less than 10% of the GFP-positive
population (ranging from three to a few dozen individual RGCs scattered over
the retina) (Fig. 3C,D). The
few remaining RGCs send out axons, projecting in a straight course to the
tectum and reach the target before and around 3 dpf, the same time as wild
type (Fig. 3A,B). We asked if
the retinotopic map was intact, despite this dramatic depletion of RGCs.
Following injection of DiI into the nasal retina and DiO into the temporal
retina of an aldehyde-fixed wild-type larva (3 dpf), we observed that nasal
axons invariably projected to the posterior tectum and temporal axons to the
anterior tectum (Fig. 3E), as
reported before (Stuermer,
1988; Baier et al.,
1996). Perhaps surprisingly, given the sparse filling of the
tectum, this topographic relationship is retained in ddl mutants
(Fig. 3F).

To test whether only the Brn3c-expressing subpopulation was reduced, we
stained the 3 dpf retina with zn5 and HuC antibodies, which label all RGCs
(zn5) or RGCs and amacrine cells (HuC). This experiment showed that most RGCs
are absent in ddl mutants (Fig.
3G-J). Amacrine cells are also reduced in number (data not shown).
DAPI staining further suggested that photoreceptors are undifferentiated, but
overall retinal lamination is unaffected
(Fig. 3K,L). The ciliary margin
of this mutant is enlarged (Fig.
3I-L) and anti-phospho-histone H3 labeling demonstrated an
increase in the number of mitotically active progenitors in this region
(Fig. 3M,N). At 3 dpf,
ddl mutants have normal body morphology and are indistinguishable
from their wild-type siblings, except for their retina phenotype. After 3 dpf,
the eye is visibly smaller, and the swimbladder fails to inflate. At about 3.5
dpf, the hearts of ddl mutants begin to show arrhythmia, suggestive
of a conductivity defect. At 4 dpf, mutants can be sorted based on pericardial
edema; they die at around 6 dpf.

daredevil (ddl) mutants have fewer RGCs. Analysis of
cell-type markers in wild type (A,C,E,G,I,K,M) and ddl
(B,D,F,H,J,L,N). (A,B) Lateral views of 78 hpf tecta, labeled with whole-mount
anti-GFP. Broken lines outline boundaries of the tectal neuropil. The
wild-type tectum (A) is covered by axons. Few axons can be detected in the
ddl tectum (B). (C,D) Confocal images of retinas in live 60 hpf
embryos. The number of GFP-positive RGCs is greatly reduced in ddl
(D). (E,F) Analysis of the retinotopic map in 72 hpf wild type (E) and
ddl (F). DiI (red) and DiO (green) were pressure injected into nasal
and temporal retina, respectively (see inserts for illustration of retinal
injection sites). The gross topography of axon targeting in ddl
mutants is not affected, with nasal axons still projecting to the posterior
tectum and temporal axons to the anterior tectum (F). (G-J) Whole-mount Zn5
staining of 72 hpf retinas. (G,H) Lateral views. (I,J) Dorsal views. The
number of zn5-positive RGCs is greatly reduced in ddl. (K,L) Sections
of 78 hpf retinae, labeled with anti-GFP (green) and DAPI (blue). The ciliary
margin (between arrowheads) in the ddl retina is wider than in wild
type. (M,N) Dorsal views of 72 hpf whole-mount retinae, labeled with
anti-phosphohistone H3 (H3P). The number of dividing cells (arrows) is greatly
increased at the ciliary margin in ddl. Scale bars: 50 μm in A and
E (for tectum panels); 20 μm in G (for retina panels).

Innervation of the tectum is delayed in vertigo
(vrt), tarde demais (tard), and late
bloomer (late) mutants

At 72 hpf, RGC axons already occupy the entire tectal neuropil. However,
the tectum is immature at this stage and retinorecipient layers are not
detectable. Large growth cones are seen, and axon arbors appear to spread out
without the characteristic adhesion between branches, which is seen later on
(see below). Three mutants, vrts1614, tards587
and lates551, were found to have few if any arbors in the
tectum at 3 dpf, although tectal architecture (as judged by DAPI histology)
appears normal. Strikingly, the retinotectal projection in these mutants
recovers later on.

vrt mutants exhibit the most severe delay. Once reaching the
boundary of the tectum, RGC axons stall
(Fig. 4A,B). After 4 dpf, the
axons resume their growth into the tectum, which they eventually cover
completely (Fig. 4C-F). Prior
to 3 dpf, RGC differentiation and axon pathfinding are normal and on schedule
(Fig. 4G,H). The number of
RGCs, expressing GFP or stained with the zn5 antibody, is identical to wild
type, and their axons follow the normal path across the midline and towards
the tectum. This suggests that the vrt mutation specifically impairs
one particular stage of axon growth: the entry into the tectum. Interestingly,
the optic tract is noticeably wider in vrt mutants. The thickening of
the optic tract could be the result of reduced fasciculation or of excessive
backbranching by axons that are stalled at the anterior pole of the tectum.
vrt mutants lack a swimbladder and die at around 10 dpf.

vertigo (vrt) mutants show severely delayed innervation
of the tectum. Analysis of retinal axon projection in wild type (A,C,E,G) and
vrt mutants (B,D,F,H). (A-F) Lateral views of the retinotectal
projection in Brn3c:mGFP transgenic fish, labeled with anti-GFP. At 3
dpf, the wild-type tectum (A) is fully innervated, while the vrt
tectum (B) is devoid of axons. Broken lines outline the tectum boundaries. In
4 dpf wild-type larvae (C), the density of axon arbors is increased compared
with 3 dpf (A), and dorsal and ventral branches are clearly visible in the
optic tract. In vrt (D), axons have invaded the anterior tectum, and
the optic tract (OT) is abnormally wide (arrowheads). At 6 dpf, axons
innervate the whole tectum in the vrt mutant (F) similar to wild type
(E) The optic tract remains wider than normal. (G,H) Dorsal views of 48 hpf
retinas and optic nerves, labeled with zn5. The number of RGCs and their axons
is similar between wild type (G) and vrt (H). Scale bars: 50μ
m.

tard and late mutants display a milder delay of the
retinotectal projection than vrt. At 3 dpf, axon arbors can be
observed in anterior tectum, but not in posterior tectum
(Fig. 5A-C). The optic tracts
are morphologically normal at all times. RGC axon growth recovers by 4 dpf,
and at 6 dpf the tectum is completely covered by fibers
(Fig. 5D-F). The tectal
neuropil in tard mutants retains an abnormal shape and its margins
are less delineated than in wild type (Fig.
5E). tard mutants fail to inflate their swim bladders and
die at around 2 weeks of age. By contrast, late mutants are
morphologically inconspicuous, including their tectum
(Fig. 5F), have swim bladders
and are adult viable. In fact, they can only be distinguished from their
wild-type siblings by the delayed innervation of the tectum around 3 dpf.

Lamination of retinal input is disrupted in dragnet
(drg) mutants

We identified two alleles, drgs510 and
drgs530, of a gene important for targeting retinal axons
to the appropriate tectal layer. drgs530 results in a
somewhat weaker phenotype than drgs510. In wild-type
zebrafish larvae, most axons extend directly into a specific layer at the
entrance of the tectum and remain confined to the chosen layer (data not
shown). Brn3c:mGFP-labeled RGCs project mostly to the two deep
sublaminae of the SFGS, and the SO is only lightly labeled (see
Fig. 2F,G;
Fig. 6A,C). In drg
mutants, this pattern of laminar targeting is perturbed
(Fig. 6B,D). Ectopic fascicles,
2-4 μm in diameter, are observed in the SO, apparently displaced from the
SFGS. GFP-labeled axons travel between the two layers. The entire tectum is
innervated in this mutant, and both tectal cytoarchitecture (by DAPI
histology; data not shown) and retinotopic mapping are intact
(Fig. 6E,F). The lamination
defects are detectable as early as 3.5 dpf and are specific to the tectum.
Sublaminar targeting of amacrine processes and of RGC dendrites in the inner
plexiform layer of the retina is normal, as shown by anti-parvalbumin and
anti-GFP double-labeling (Fig.
6G-N). The only other detectable phenotype of drg mutants
is an opaque lens after 5 dpf, caused by overgrowth by epithelial cells
(Fig. 6G,H). drg
mutants are fully viable as adults.

In fuzzs531, beyos578 and
breks574 mutants, the retinotectal projection forms on
time and is initially indistinguishable from wild type, but the density of
axon arbors is reduced after 5 dpf. Optic tracts are of normal width and show
the characteristic branching pattern, as the RGC fibers enter the tectum. This
suggests that the normal complement of axons is present, but that their arbors
are smaller or have fewer branches. The borders of the retinotectal fiber zone
are less well demarcated (`fuzzy') in these mutants, with axons and growth
cones frequently straying outside the neuropil area
(Fig. 7). Tectal
cytoarchitecture is unaltered, as determined by DAPI staining. In
brek mutants, the GFP label in the tectal neuropil becomes punctate
after 4 dpf, sometimes as late as 6 dpf, indicating that axons are
disintegrating in this mutant (see Fig. S1 in the supplementary material). In
a substantial proportion of fish, axon degeneration is uneven, with one region
of the tectum initially more affected than another, although a time-course
analysis showed that the temporal pattern is inconsistent between animals
(n=6). We could not detect an excessive number of TUNEL-positive
cells in the mutant retina at 5 dpf, suggesting that axon retraction is not
secondary to RGC death in the retina (data not shown). The axon degeneration
phenotype is not seen in fuzz or beyo. In beyo
mutants, the inner plexiform layer of the retina is misshaped at the margins,
bending distally to the outer nuclear layer (see Fig. S2 in the supplementary
material), and the telencephalon is reduced (data not shown). All three
mutants do not inflate their swimbladders and die as young larvae.

tarde demais (tard) and late bloomer
(late) mutants show mildly delayed innervation of the tectum. (A-F)
Confocal images of Brn3c:mGFP labeled retinotectal projections of 80
hpf tecta in live larvae (A-C). The wild-type tectum (A) is filled with
retinal axons. The optic tract has branched into stereotyped fascicles,
labeled with numbers. The tard tectum (B) and the late
tectum (C) are less than halfway innervated. (D-F) Lateral views of 6 dpf
tecta. The tard tectum and the late tectum are now covered
with axons. However, the tard tectum is small and its boundary
remains abnormal (E), compared with wild type (D) and in contrast to
late (F). Asterisks indicate melanophores on the skin. Broken lines
outline the tectal neuropil. Scale bars: 20 μm.

In wild type, axons are sorted in the optic tract according to their
topographic position and enter the tectum through a set of stereotyped
fascicles (Stuermer, 1988)
(see Fig. 5C). After entering
the tectum, axon arbors do not spread uniformly, but rather form a
characteristic grid within the neuropil, with regions of high fiber density
evidently separated by gaps. Thick fascicles circle the perimeter of the
tectum, from which single axons depart at various positions to navigate to
their respective targets in the center of the neuropil. Thin fascicles are
also observed traveling through the wild-type tectum. This organization
becomes evident in confocal images of Brn3c:mGFP tecta at high
magnification (Fig. 8A,B).

Tectal neuropil boundaries dissolve in fuzz wuzzy (fuzz),
beyond borders (beyo) and breaking up
(brek) mutants. (A-D) Z-projections of confocal image stacks
showing lateral views of tecta in 6 dpf live Brn3c:mGFP larvae. The
boundary of the tectum is smooth and well defined in wild type (A). In
fuzz (B), axon arbors are less dense in the tectal neuropil and often
overshoot the tectal boundary (arrowheads). The overshooting phenotype is more
severe in beyo (C) and brek (D). Asterisks indicate
melanophores. Scale bars: 20 μm.

In a heterogeneous group of four mutants, clews567,
comas532, bluks582 and
blins573, the RGC fibers are more diffuse and less
mesh-like than in wild type (Fig.
8C,D), or appear to meander in the neuropil
(Fig. 8E,F). We interpret the
`diffuse' phenotype as a lack of fiber-fiber adhesion, but the apparent
dispersion may have other causes as well. Although tectal cytoarchitecture
appears overall normal (by DAPI histology), the tectum is slightly smaller in
coma and bluk. In coma mutants, the optic tract is
already disorganized upon entering the tectum: axons preferentially join the
dorsal and ventral branches and appear to avoid the more centrally positioned
fascicles (Fig. 8F). The
fascicles circling the tectum are also thicker and more compact. However, DiI
and DiO double-labeling of RGC axons originating from the nasoventral and
temporodorsal quadrants, respectively, demonstrated that the retinotopic map
is intact (data not shown). The coma mutant also has a specific
retinal defect: a small number of the newborn RGCs at the ciliary margin are
mispositioned in the distal retina near the inner nuclear layer. These cells
express GFP at the same intensity as regularly positioned RGCs and exhibit a
neuronal morphology (see Fig. S2 in the supplementary material). The
coma and bluk mutations are larval lethal, while
blin and clew mutants are adult viable.

Discussion

We generated a transgenic zebrafish line that expresses membrane-targeted
GFP under control of a brn3c enhancer fragment. Brn3c is a POU-domain
transcription factor, which is involved in RGC differentiation and axon
outgrowth (Liu et al., 2000;
Wang et al., 2002). The
Brn3c:mGFP transgene is expressed in a subset of RGCs and in
mechanosensory hair cells, similar to endogenous brn3c
(DeCarvalho et al., 2004;
Erkman et al., 1996;
Xiang et al., 1995). The
optical transparency of zebrafish embryos and larvae allowed us to visualize
the retinotectal projection in living fish, relying solely on the crisp
labeling of RGC axons by membrane-bound GFP. We could thus investigate, at
unprecedented resolution, tectal neuropil organization, layer formation,
fasciculation and the time course of innervation in both wild-type and mutant
zebrafish. A screen of 168 ENU-mutagenized genomes in the Brn3c:mGFP
background revealed 13 mutations disrupting the orderly innervation of the
tectum (Fig. 9). The affected
genes may be expressed in the RGCs or the tectum, or both. The newly
discovered mutant phenotypes can be categorized into five groups
(Table 1), none of which has,
to our knowledge, been described before.

We designed our new GFP-based screen to find phenotypes that would have
escaped discovery in the earlier screen
(Karlstrom et al., 1996;
Trowe et al., 1996) or would
have been difficult to analyze in sufficient detail using carbocyanine tracing
alone (Burrill and Easter,
1994; Kaethner and Stuermer,
1992; Stuermer,
1988). We concentrated on mutations disrupting the fine
architecture and temporal coordination of the retinotectal projection, taking
advantage of the highly reproducible label afforded by a genetically encoded
reporter, by its stability, and by its cell-type specific expression pattern.
Although the scale of our new screen was less than one-tenth of the original
retinotectal screen, it was very productive in detecting specific phenotypes.
The previous screen had enriched for mutants with pleiotropic phenotypes. As a
result, of the 114 mutants described, all but eight (7%) die as young larvae,
indicating pervasive, non-visual defects. For comparison, of the 13 new
mutants, five are adult viable (39%) and most of them show few if any
phenotypes outside the visual system (Table
1).

Summary of retinotectal mutants. The 13 loci are predicted to affect
different aspects of tectal development and architecture. The ddl
protein is required for differentiation of most RGCs. The vrt, tard
and late gene products ensure timely innervation of the tectum.
Products of vrt and coma organize the fascicles in the optic
tract, while bluk, clew, blin and coma regulate fiber-fiber
interaction in the tectum. Proteins encoded by fuzz, beyo and
brek confine axons to the neuropil, while drg is required
for targeting axons to the SFGS. (The deeper layers of the tectum, SGC and
SAC/SPV, are not labeled by Brn3c:mGFP.)

Our screen was preordained to find mutations disrupting RGC
differentiation, as these are sure to prevent, or perturb, axonal projections
to the tectum. In fact, one mutant (ddl) with sparse innervation of
the tectum, was shown to produce only a fraction (less than 10%) of the normal
complement of RGCs. The transcription factor Brn3b is required for
differentiation, pathfinding and survival of RGCs, and might therefore be a
candidate gene for ddl (Erkman et
al., 2000; Liu et al.,
2000). However, the few remaining RGCs, which are spared in
ddl mutants, are able to extend axons into the tectum in a straight
course, suggesting that pathfinding is normal. Moreover, other organs, such as
the heart, are also affected, arguing against brn3b as a candidate
gene. ddl is also distinct from lakritz/ath5, whose mutation
leads to a complete loss of RGCs, but otherwise milder retinal defects
(Kay et al., 2001;
Kay et al., 2004). The unique
combination of phenotypes, together with its genetic map position (T.X.,
unpublished), suggests that ddl encodes a gene not previously
implicated in RGC development.

In wild type, the first RGC axons enter the tectum at 48 hpf and reach its
posterior end within the next 24 hours
(Stuermer, 1988). The larvae
display their first visual reflexes at around the same time, suggesting that
synaptogenesis is rapid (Easter and
Nicola, 1996). We discovered three genes, vrt, tard and
late, important for keeping innervation of the tectum on this tight
schedule. In these mutants, axons stall at the anterior end of the tectum and
invade it after a substantial delay. The mutations may disrupt extracellular
signals that regulate innervation of the tectum, or a component of the
transduction machinery that transmits these signals inside the growth cone and
links them to the cytoskeleton. Blocking FGF signaling in the Xenopus
retinotectal projection results in a bypass phenotype, where RGC axons grow
around the tectum (McFarlane et al.,
1996; Walz et al.,
1997). This is different from the stalled growth we observed here
in zebrafish vrt mutants. Alternatively, one or more of these genes
may encode a factor required for axon elongation, such as a cytoskeletal or
motor protein. Strikingly, the defects are only transient - by 6 dpf, the
retinotectal projection has recovered - and appear to be specific to the optic
tract/tectum boundary along the visual pathway. Although a retinotectal map
forms eventually, these mutants remain completely (vrt) or partially
blind (tard, late) (T.X., unpublished). It will be interesting to
find out if these persistent visual impairments are secondary to the delayed
innervation or caused by a direct effect on visual functions by the respective
gene products.

Axon-axon interactions are important for assembling a highly organized
neural structure and for maintaining its integrity. As the optic tract merges
with the anterior border of the tectal neuropil, retinal axons are ordered
into a stereotyped set of fascicles (von
Bartheld and Meyer, 1987). About ten thin fascicles emanate from
two thicker branches, the dorsal (medial) and the ventral (lateral) brachia of
the optic tract, which mainly contain axons of ventral and dorsal RGCs,
respectively. These fascicles continue their course within the tectal neuropil
and around its margins. Axons leaving these fascicles branch to form arbors
near their termination zone. Confocal analysis further demonstrates that fiber
distribution in the neuropil is not homogeneous, suggesting that adhesion
between branches of neighboring arbors produces a fine-meshed grid. Thus,
Brn3c:mGFP labeling showed an intricate organization of axons and
arbors in the wild-type tectum, which, to our knowledge, has not been the
subject of previous analysis and whose functional significance is unknown.

Our screen discovered two mutants with optic tract phenotypes, clearly
different from box and dak
(Lee et al., 2004), and four
with putative neuropil adhesion phenotypes. In vrt mutants, RGC axons
appear diffuse and invade the tectum in several broad bundles. In
coma mutants, the central-most fascicles are depleted of axons, and
the marginal fascicles, at the dorsal and ventral edges of the optic tract,
are expanded instead. Neuropil organization is also disrupted in coma
mutants, where most axons are bundled around the margins of the neuropil and
the few internal fascicles are seen meandering within the neuropil. In
bluk, blin and clew mutants, the regularly spaced axon
meshwork in the tectum is diffuse. The corresponding genes may encode adhesion
molecules or factors that influence branching. For example, cadherins
(Elul et al., 2003;
Inoue and Sanes, 1997;
Liu et al., 1999;
Miskevich et al., 1998;
Riehl et al., 1996;
Stone and Sakaguchi, 1996;
Treubert-Zimmermann et al.,
2002) and immunoglobulin superfamily molecules, such as L1 and
NCAM (Lyckman et al., 2000;
Rathjen et al., 1987;
Thanos et al., 1984;
Vielmetter et al., 1991;
Yamagata et al., 1995), are
expressed by both RGC axons and their targets in a variety of vertebrates and
could therefore underlie some of the recognition events that underlie fascicle
formation, axon arbor organization or synapse stabilization.

Many areas in the vertebrate CNS are layered, and laminar targeting by
axons is thought to be crucial for synaptic specificity
(Sanes and Yamagata, 1999).
Although a DiI tracing study in larval zebrafish made only cursory mention of
tectal layers (Burrill and Easter,
1994), our confocal analysis of the Brn3c:mGFP and the
Shh:GFP transgenic lines demonstrates that exactly four well-defined
retinorecipient layers exist at 6 dpf. In adult teleost species, four major
layers of retinal fibers have been described
(Reperant and Lemire, 1976;
Vanegas and Ito, 1983;
von Bartheld and Meyer, 1987),
with very similar characteristics to the layers we saw in the larvae. We have
therefore adopted the nomenclature developed for the adult structures. Only
the SFGS, receives substantial input from Brn3c:mGFP-expressing RGCs,
and not all the axons that innervate these layers are GFP positive. Retinal
axons in the SO and in the two deeper layers, SGC and SAC/SPV, are labeled in
Shh:GFP fish and by whole-eye DiI fills, but not at all (SGC,
SAC/SPV) or very little (SO) in Brn3c:mGFP fish. Brn3c:mGFP
provides one of the first examples of a marker differentially expressed among
RGCs with different laminar specificity.

The layer-specificity of retinal afferents is formed and maintained by
molecular cues in the tectum, such as N-cadherin
(Inoue and Sanes, 1997;
Miskevich et al., 1998;
Yamagata et al., 1995),
DM-GRASP (Yamagata et al.,
1995), neuropilin (Feiner et
al., 1997; Takagi et al.,
1995) and Ephrin B molecules
(Braisted et al., 1997).
Furthermore, patterning genes such pax7
(Thompson et al., 2004) and
grg4 (Nakamura and Sugiyama,
2004) may have a role in patterning the layers of the mammalian
superior colliculus. We identified an apparently novel gene, drg,
which is important for specifying SFGS and SO layer identities. In
drg mutants, retinal axons travel between the SO and SFGS, often in
thick fascicles. The SO appears more extensively innervated. These
observations suggest that axons that would normally project to the SFGS are
misrouted to the SO. The drg gene might encode an adhesion molecule
expressed in the SFGS, or a repellent factor present in the SO. Alternatively,
the mutation may disrupt transduction of layer-specific cues inside retinal
growth cones. The drg mutant is viable and therefore offers the
opportunity to study the fate of the mistargeted axons in later life.

Although a great deal is known about molecules that guide axons to the
tectum and align their arbors retinotopically within it, it is not clear what
confines them to the tectal neuropil. In wild type, many retinal axons grow
around the tectum in thick bundles, from which individual axons or small
fascicles depart into the center of the neuropil. The Brn3c:mGFP
labeling allowed us to identify three mutants, fuzz, beyo and
brek, in which this organization has broken down. The neuropil edges
are less well demarcated, and axons overshoot the borders. This description is
superficially reminiscent of the zebrafish acerebellar/fgf8
(ace) mutant, which lacks the midbrain-hindbrain boundary and has an
enlarged tectum (Jaszai et al.,
2003; Picker et al.,
1999), although the phenotypes of our new mutants do not match the
comparatively severe defects of ace. Repellent factors, such as
Ephrin A3 (Hirate et al.,
2001), Ephrin B2a (Wagle et
al., 2004) and Tenascin (Becker
et al., 2003; Perez and
Halfter, 1994; Yamagata et
al., 1995), as well as adhesion molecules
(Demyanenko and Maness, 2003),
may play a role in demarcating neuropil boundaries, so these genes represent
plausible candidates for fuzz, beyo and brek.

Our study demonstrates the feasibility of a sensitive screen for subtle
anatomical disruptions in the vertebrate brain. The genetically encoded GFP
reporter provides resolution superior to carbocyanine dyes and pan-RGC
markers. Our small-scale effort helped to discover larval- and adult-viable
zebrafish mutants with changes in the patterning of retinotectal connections.
Using Brn3c:mGFP, or similar lines, it should be possible to perform
a saturated screen for genes assembling the optic tectum and other areas of
the vertebrate brain.

Supplementary material

Acknowledgments

We thank Pamela Raymond, Ann Wehman, Matthew Smear, and Linda Nevin for
comments on the manuscript and all members of our laboratory for advice.
Chi-Bin Chien (MPI Tübingen/University of Utah) provided pG1, the
original GFP vector. This work was supported by a fellowship from the UCSF
Neuroscience Training grant (T.X.), by the David and Lucile Packard Foundation
and by grants from the NIH Eye Institute (H.B.).

Meek, J. (1983). Functional anatomy of the
tectum mesencephali of the goldfish. An explorative analysis of the functional
implications of the laminar structural organization of the tectum.
Brain Res.287,247
-297.

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